The ultimate goal of this project is to understand the molecular mechanisms of protein synthesis. The wealth of structural information obtained from cryoEM reconstructions and x-ray crystallography in recent years has provided a strong basis for studying the molecular basis of ribosome function and has led to proposal of numerous mechanistic models. However, it is essential to subject such structure-based models to experimental test. Moreover, the realization that ribosomes are highly dynamic structures, whose functional capabilities are based on both large- and small-scale molecular movements, requires experimental approaches that will be able to correlate specific dynamic events with function. This project will investigate the mechanisms of crucial steps in translation, in particulr, steps where ribosome structural dynamics play an important role and including experiments specifically designed to critically test mechanisms based on structural observations. The experimental approaches are based on biochemical and biophysical solution methods, including in vitro testing of specific functions in mutationally altered ribosomes, bulk and single-molecule FRET approaches and the use of optical tweezers. The principle aims are to investigate (i) the molecular basis of ribosomal translocation; (ii) the mechanism of translation termination; and (iii the ribosomal mRNA helicase and measurement of ribosomal forces using optical tweezers. The proposed research is of strong clinical relevance because bacterial ribosomes are a major target for numerous antibiotics. Elucidation of the structures of functional complexes of bacterial ribosomes will provide a rigorous basis for development of novel antibiotics to address the crisis of emerging drug-resistant strains of bacterial pathogens. Studying the mechanism of action of the ribosomal helicase will help to understand how retroviruses, including HIV, use pseudoknots to create programmed frame-shifting during their infectious cycle, providing clues to the design of anti-retroviral therapeutics. PUBLIC HEALTH RELEVANCE: This research is of strong clinical relevance because bacterial ribosomes are a major target for numerous antibiotics. Elucidation of the mechanisms of action of bacterial ribosomes, together with knowledge of their high-resolution structures, will therefore provide a rigorous basis for development of novel antibiotics to address the crisis of emerging drug-resistant strains of bacterial pathogens. Studies on the mechanism of action of the ribosomal helicase will help to understand how retroviruses, including HIV, use pseudoknots to create programmed frame-shifting during their infectious cycle, providing clues to the design of anti-retroviral therapeutics.